Cardiac stem cell genetic engineering using the alphaMHC promoter

Abstract

Aims: Cardiac stem cells (CSCs) show potential as a cellular therapeutic approach to blunt tissue damage and facilitate reparative and regenerative processes after myocardial infarction. Despite multiple published reports of improvement, functional benefits remain modest using normal stem cells delivered by adoptive transfer into damaged myocardium. The goal of this study is to enhance survival and proliferation of CSCs that have undergone lineage commitment in early phases as evidenced by expression of proteins driven by the alpha-myosin heavy chain (alphaMHC) promoter. The early increased expression of survival kinases augments expansion of the cardiogenic CSC pool and subsequent daughter progeny.

Materials & methods: Normal CSCs engineered with fluorescent reporter protein constructs under control of the alphaMHC promoter show transgene protein expression, confirming activity of the promoter in CSCs. Cultured CSCs from both nontransgenic and cardiac-specific transgenic mice expressing survival kinases driven by the alphaMHC promoter were analyzed to characterize transgene expression following treatments to promote differentiation in culture.

Results & conclusion: Therapeutic genes controlled by the alphaMHC promoter can be engineered into and expressed in CSCs and cardiomyocyte progeny with the goal of improving the efficacy of cardiac stem cell therapy.

Figure 1. Cardiac stem cells from transgenic mice express αMHC driven transgenes in vivo and in culture

(A) Immunolabeling of adult _MHCGFP mouse heart section shows colocalization of c-kit (red) and GFP (green) staining. Desmin (blue) also colocalizes with GFP in mature myocytes. Scale bar is 10 μM. (B) CSCs isolated from _MHCGFP, _MHCAkt-nuc and _MHCPim-wt were cultured in growth media and harvested for RNA isolation and cDNA synthesis. cDNA was amplified using primers specific for the transgene inserted into each CSC line. (C) Flow cytometric analysis of NTG, _MHCPim-wt and _MHCAkt-nuc CSCs either untreated (left) or hydrogen peroxide treated (right) stained for annexin V and 7-AAD apoptosis markers (representative of n = 2 independent experiments). (d) Fold-increase quantitation of annexin V⁺/7-AAD⁺ CSCs after hydrogen peroxide treatment in (C). (e) Trypan blue exclusion hemocytometer counts of NTG, _MHCPim-wt and _MHCAkt-nuc CSCs seeded at equal amounts on day 0 and counted 2 or 4 days later (representative of n = 3 independent experiments). 7-AAD: 7-amino-actinomycin D; CSC: Cardiac stem cell; GFP: Enhanced green fluorescent protein; MHC: Myosin heavy chain; NTG: Nontransgenic.

Figure 2. Nontransgenic cardiac stem cells express tagged fluorescent marker proteins upon transfection with plasmid constructs under the control of the αMHC promoter

Nontransgenic cardiac stem cells were cultured in growth media, transfected with plasmid constructs, allowed to express for 48 h and fixed. (A) CMV-Td-Tomato-Flag construct (left). Cells were transfected with a Tomato-Flag construct under the viral CMV promoter as a control and stained with c-kit or Flag-tag (green) and Topro (blue), td-Tomato protein fluorescence (red) (right). (B) αMHC-YFP-HA construct (left). Cells were transfected with a YFP-HA construct under the αMHC promoter and stained with c-kit or HA (red), Topro (blue) and YFP fluorescence (green) (right). (C) Diagram of αMHC-GFP construct (left). Cells were transfected with a GFP construct under control of the αMHC promoter and stained with c-kit (red), Topro (blue) and GFP protein fluorescence (green) (right). Scale bar is 40 μm. CMV: Cytomegalovirus; eGFP: Enhanced Green fluorescent protein; MHC: Myosin heavy chain; YFP: Yellow fluorescent protein.

Figure 3. Cardiac stem cells express transgenes controlled by the αMHC promoter in the appropriate subcellular localization in culture by confocal microscopy

(A) NTG and (B) _MHCPim-wt CSCs cultured in growth media, fixed and immunolabeled with antibodies to Pim (green), c-kit (red) and Topro (blue). _MHCPim-wt CSCs show increased Pim expression localized to the cytoplasm. (C) NTG and (d) _MHCAkt-nuc CSCs cultured in growth media, fixed and immunolabeled with Myc-tag (green), c-kit (red) and Topro (blue). _MHCAkt-nuc CSCs show nuclear-localized myc-tag expression. Scale bar is 40 μm. CSC: Cardiac stem cell; MHC: Myosin heavy chain; NTG: Nontransgenic.

Figure 4. Cardiomyogenic differentiation allows for persistent αMHC driven transgene expression

(A) _MHCPim-wt and (B) NTG CSCs differentiated on fixed NRCMs for 7 days, fixed and immunolabeled for Pim (green), α-sarcomeric actinin (red) and Topro (blue). _MHCPim-wt differentiated CSCs express more Pim protein. (C) _MHCPim-wt and (D) NTG CSCs differentiated on fixed NRCMs for 7 days, fixed and immunolabeled for GFP (green), α-sarcomeric actinin (red) and Topro (blue). _MHCPim-wt differentiated CSCs express GFP. (E) _MHCAkt-nuc and (F) NTG CSCs differentiated on fixed NRCMs for 7 days, fixed and immunolabeled for myc-tag (green), α-sarcomeric actinin (red) and Topro (blue). _MHCAkt-nuc differentiated CSCs express increased nuclear-localized myc-tag. Scale bar is 40 μm. (G) RT-qPCR analysis of NTG CSCs postdifferentiation on live NRCMs shows an increase in relative mRNA levels of troponin T (left), tropomyosin (middle) and α-smooth muscle actin (right) compared with undifferentiated CSCs. CSC: Cardiac stem cell; GFP: Green fluorescent protein; MHC: Myosin heavy chain; NRCM: Neonatal rat cardiomyocyte; NTG: Nontransgenic.

Figure 5. Differentiation of cardiac stem cells by exposure to dexamethasone results in loss of transgene expression

(A) _MHCPim-wt CSCs cultured in growth media, fixed and immunolabeled for c-kit (green), Pim (red) and Topro (blue). (B) _MHCPim-wt CSCs treated with dexamethasone containing media for 7 days, fixed and immunolabeled for c-kit (green), Pim (red) and Topro (blue) show decreased c-kit as well as Pim expression. (C) _MHCAkt-nuc CSCs cultured in growth media, fixed and immunolabeled for c-kit (green), myc-tag (red) and Topro (blue). (D) _MHCAkt-nuc CSCs treated with dexamethasone containing media for 7 days, fixed and immunolabeled for c-kit (green), Myc-tag (red) and Topro (blue) show decreased c-kit as well as myc-tag expression. Scale bar is 100 μm. CSC: Cardiac stem cell.